The present invention relates to a composition of matter in which microspheres are covalently bonded to a support of solid substrate, the thus-bonded microspheres containing residual reactive functions.
Due to their spherical shape and high surface area, microspheres have numerous applications such as specific cell labelling, cell separation, phagocytosis, diagnostics, cell growth, affinity chromatography and hemoperfusion (see e.g., Margel, S., Applied Biochemistry and Biotechnology, 1983, 8, 523; Lazar, A., Silverstein, L., Margel, S. and Mizrahi, M., Dev. in Biol. Stand., 1985, 60, 456; Pines, M. and Margel, S., J. of Immunoassay, 1986, 7, 97; Palzer, R., Walton, J. and Rembaum, A., In Vitro, 1978, 14, 336; Rembaum, A. Yen, S. P. S. and Volkson, W., Chem. Tech., 1978, 8, 182). Recently, in order to improve the quality and usefulness of polymeric microspheres, significant progress has been made in the synthesis of microspheres with narrow size distribution. Highly uniform polymeric microspheres are currently effective for applications such as adsorbents for HPLC, calibration standards and spacers for liquid crystals [Ugelstad, J., Soderberg, L., Berge, A. and Bergstom, J., Nature (London), 1983, 303, 5].
From a practical point of view, the efficiency and use of polymeric microspheres in solution, particularly microspheres smaller in their diameter than approximately 0.4.mu., are still limited, because of some major disadvantages, e.g., difficulties in separation of free ligand from ligand bonded to the microspheres and instability of the microspheres in solution towards agglutination. The latter disadvantage is the major reason for the difficulties obtaining while carrying out reactions with polymeric microspheres.
Polymeric microspheres covalently bonded with appropriate antibodies or lectins have been studied for mapping of cell receptors. For example, polyaldehyde microspheres in sizes ranging from 0.1.mu. to 0.7.mu. bonded with anti-thy 1,2 antibodies were used for specific labelling of T lymphocytes. Similar microspheres covalently bonded with the drug disodium chromoglycate were used for specific labelling of rat basophilic leukemia cells (Pacht, I., Mazurek, N. and Margel, S., Drug Conjugates of Polymeric Microspheres as Tools in Cell Biology, Plenum Publishing Corporation, 1982, pp. 109-123).
Polystyrene beads crosslinked with divinylbenzene of approximately 30.mu. diameter containing on the surface sulfate groups (negative charge) electrostatically attached to polystyrene particles of 0.1-0.5.mu. diameter containing quaternary ammonium groups (and which are available from Dionex Corporation), are used in ion chromatography for ion separation (Small, H., Stevens, T. S. and Bauman, W. W., Anal. Chem., 1975, 47, 1801; Gjesde, D. T. and Fritz, J. S., in "Ion Chromatography", 2nd edn., Huhig, Heidelberg, 1987). These hybrid type ion exchange resins (pellicular resins) have a low capacity; moreover, the attached colloid particles are limited in their size (up to approximately 0.5.mu.) and can be removed from the bead core by competition reaction.
A few patents describe the adhesion of various microsphere types, e.g. glass microspheres, to an appropriate support, by impregnating the latter with the microspheres in the presence of a thickener and an adhesive agent for binding the microspheres to the support. This binding agent usually contains epoxide compounds or other adhesive materials (Seuzaret, L., FR 2,609,835; Thomson, E. et al EP 209337; Hicks, I. A. et al, U.S. Pat. No. 4,548,863).
The patents literature also describes a procedure for grafting of polyacrolein microspheres having diameters of to 0.2.mu. onto the surface of organic polymers such as polystyrene, by high energy radiation (Co irradiation) process (Margel, S., IL 67619; Rembaum, A. et al, U.S. Pat. No. 4,534,996). According to this process, a deaerated acrolein in aqueous solution is polymerized in the presence of an appropriate surfactant and the organic support, by the high energy source. However, this method suffers from a number of major disadvantages: (a), the need for a high energy source; (b) the mechanism for the high energy process is not clear and the process is not properly controllable; (c) the structure of the obtained composite materials is not homogeneous and distinctive, and furthermore, part of the surface of the grafted support is barely coated with microspheres, while the other part of the surface is coated heterogeneously with one or several layers of microspheres; (d) the high energy process for covalent binding of polyacrolein microspheres onto the surface of polymers is not applicable to inorganic substrates, e.g. glass, semiconductive materials such as silicon, and some organic polymers such as polytetrafluoroethylene; (e) the high energy process is not applicable for binding onto the surface of solid substrates, microspheres which were previously prepared.
The present invention relates to compositions of matter which incorporate microspheres, while avoiding the disadvantages of the prior art products.